Hypokalaemic periodic paralysis and myotonia in a patient with homozygous mutation p.R1451L in NaV1.4

Figures

• 

  Figure 1. The Pedigree of Family 1. Co-segregation analysis revealed that the proband carried a homozygous p.R1451L mutation in NaV1.4, while the consanguineous parents and the sister carried the variant in heterozygosis.

• 

  Figure 2. Electrodiagnostic studies of homozygous and heterozygous p.R1451L carriers. (A) A significant CMAP amplitude decline was demonstrated in the homozygous patient (left). An increase of baseline amplitude was demonstrated for the proband after Coenzyme Q10 treatment (right). (B) A significant CMAP amplitude decline was demonstrated in the father and the sister, but not the mother.

• 

  Figure 3. NaV1.4 R1451L does not conduct gating pore currents. (A) Representative current traces of wild-type and p.R1451L channels expressed in Xenopus oocytes. The response to test voltages ranging from −140 mV to +50 mV in 5 mV increments is shown. Holding voltage was −100 mV. The capacitive currents are clipped. Scale bars are 50 ms (x) and 0.5 µA (y) (B) Leak subtracted current plotted against the test voltage for wild-type (solid circles, n = 5) and p.R1451L (open circles, n = 10) channels in 50% guanidinium solution. The current of p.R1451L did not differ from the wild-type (I−80 p.R1451L 64 ± 18 nA vs. WT 58 ± 16 nA). As positive control, the current response of hypoPP mutant NaV 1.4 R222W (triangle, n = 3) is shown (I−80 = −567 ± 81 nA).

• 

  Figure 4. Voltage dependence of activation and fast inactivation. Number of experiments is given in Table 2. (A) Representative current traces of wild-type and p.R1451L channels in response test voltages ranging from −100 mV to +50 mV in 10 mV increments, followed by a tail pulse to −10 mV. Holding voltage was −100 mV. Y-axis scale bar is 20 pA/pF. The voltage protocol is shown below. (B–D) Wild-type data is shown in solid symbols, p.R1451L data in open symbols. (B) Peak current density is plotted against the corresponding test voltage. (C) The current data from A is converted to conductance. (D) Peak tail current at −10 mV is plotted against the test voltage. For (C) and (D), data from individual cells was normalised to top and bottom amplitude of a Boltzmann fit. Mean ± SEM of normalised data is shown. Solid lines represent the fit of a Boltzmann equation to the mean data.

• 

  Figure 5. Inactivation properties. Wild-type data is shown in solid symbols, p.R1451L data in open symbols. Number of experiments is given in Table 2 and voltage protocols are described in the methods and shown in (G). (A) Rate of closed state inactivation at −80 mV (grey) and −60 mV (black) from Vh −100 mV for wild-type and p.R1451L channels. Current in response to test voltage is plotted against the time at pre-pulse voltage. Data are normalised to maximum current in response to test pulse. (B) Time constant of open state inactivation at voltage range −20 mV to +20 mV. (C) Rate of recovery from inactivation at −80 mV (black) and −100 mV (grey) for wild-type and p.R1451L channels. Current in response to second test pulse is divided by current in response to first pulse and plotted against the time at recovery voltage. (A–C) Solid lines represent the fit of an exponential function to the mean data. (D) Peak current amplitude in response to 2 ms pulse to 0 mV is plotted against the pulse number when the train of pulses was applied at 40 Hz, 100 Hz and 200 Hz frequency for wild-type (5–8 cells/frequency) and p.R1451L (5–14 cells/frequency) channels. Current is normalised to the amplitude of…

• 

  Figure 6. Location of R1451 in the “up” conformation of NaV1.4 VSD-IV. In the cryo-EM structure of electric eel NaV1.4 (protein data bank ID 5XSY) the residue corresponding to NaV1.4-R1451 (blue spheres) is located in proximity to the residue corresponding to NaV1.4-E1373 (pink spheres) in the S1-S2 loop of VSD-IV. The distance between the guanidino- and delta-carbons of R1451 and E1373, respectively, is 5.78 Å. Arginines corresponding to R1448, R1454 and R1457 are shown in blue sticks. The structure is illustrated with the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.